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In the therapeutic target tissue of skeletal muscle, Vascular Endothelial Factor-A (VEGF) does not induce vascular sprouting, but rather intussusceptive angiogenesis, which is yet poorly understood. Intussusceptive morphogenesis entails: 1) circumferential enlargement; 2) formation and fusion of intraluminal endothelial pillars; 3) longitudinal splitting into new vessels. Here we sought to resolve the transient states of endothelium underlying intussusceptive angiogenesis by VEGF delivery in skeletal muscle.

Endothelial cells (ECs) were isolated from mouse limb muscles 3, 4, 5 and 7 days after delivery of a therapeutically relevant VEGF dose, and sequenced. These time-points comprise all stages of intussusception. Protein expression was verified by immunofluorescence confocal microscopy.

Sub-clustering of VEGF-activated endothelium revealed 5 individual functional communities, which were identified as: 1) Angiogenic Capillary; 2) Proliferation; 3) Migratory Vein; 4) Interferon; and 5) Endothelial-to-Mesenchymal Transition (EndoMT). The Interferon community appeared only during the resolution phase after splitting, while the others co-existed during the enlargement/pillar stages. The EndoMT signature appeared distinctive for intussusception, in comparison with a pure sprouting model in the retina. Conversely, previously published signatures failed to identify a Tip Cell community in the intussusception dataset but correctly identified the Proliferation community. Multiplex immunostaining identified Endo-MT cells co-expressing activated TGF-B signaling (pSMAD), Zeb2 transcription factor and fibronectin-1 during the enlargement/pillar stages, with gradual resolution after splitting, whereas these were absent in the retina sprouting front. Lineage tracing with Cdh5-Cre:Ai14 (tdTomato) mice showed that the vast majority of EndoMT ECs do not fully transition into mesenchymal cells. Instead, EndoMT appears to be transient and reversible, suggesting a temporary phenotypic shift that supports structural reorganization during intussusception. Ongoing experiments with EC-specific knockout of Zeb2 aim to define how loss of EndoMT influences the process of intussusception.

The angiogenic mechanisms of sprouting and intussusception are transcriptionally distinct and the EndoMT process is specific for intussusceptive angiogenesis by VEGF delivery in skeletal muscle.

Cerebrospinal fluid (CSF) outflow maintains brain homeostasis by clearing metabolic waste and regulating intracranial pressure. While arachnoid granulations were long considered the primary drainage route, recent studies show CSF also drains into the lymphatic system via the cribriform plate, perivascular spaces in the anterior and medial cranial fossa, and spinal nerve roots. These findings challenge classical models and carry important implications for diseases involving impaired fluid dynamics, including hydrocephalus, Alzheimer's disease, and traumatic brain injury.

To investigate the complexity of CSF outflow pathways in vivo and their alterations during aging and disease, we performed synchrotron radiation-based micro computed tomography (SRµCT) of CSF outflow in living mice. This approach enables high-resolution, dynamic imaging of CSF contrast agent distribution throughout the cranial central nervous system. Deeply anesthetized mice were implanted with a cannula in the lateral ventricle allowing for continuous infusion of a barium sulfate nanoparticle-based contrast agent. Data were acquired as a time series of 50 scans (at 1 scan/minute) at the Biomedical Imaging and Therapy beamline at the Canadian Light Source in Saskatoon, Canada. The acquired projections were reconstructed into a 24×16×16 mm3 volume with 8 µm voxel size, with CSF contrast agent distribution visualized using U-Net network-based deep-learning segmentation. The final visualization of the 3D rendered and segmented datasets was done using Dragonfly software.

These data show CSF exits the cranial cavity via multiple pathways—the cribriform plate to nasal and nasopharyngeal lymphatics, the anterior cranial fossa, and the jugular foramen—draining into superficial and deep cervical lymph nodes. High-resolution SRµCT allowed to follow contrast-agent distribution into the subarachnoid cisterns and dural tissue. Counter to the glymphatic model, we did not observe tracer spread into penetrating perivascular spaces or brain parenchyma.

By integrating in vivo SRµCT imaging with widefield and confocal microscopy of decalcified tissues from various reporter mouse models injected with CSF tracers, we are currently creating a comprehensive 4D atlas of CSF outflow of the entire mouse brain, mapping its dynamics down to the cellular level.